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Colloids and Surfaces B: Biointerfaces 53 (2006)

Colloids and Surfaces B: Biointerfaces 53 (2006)

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Colloids and Surfaces B: Biointerfaces 53 (2006) 193–202
Development and characterization of new insulincontaining polysaccharide nanoparticles
Bruno Sarmento
a
,
, Ant´onio Ribeiro
b
, Francisco Veiga
c
, Domingos Ferreira
a
a
 Department of Pharmaceutical Technology, Faculty of Pharmacy of the University of Porto, Rua An´ıbal Cunha 164, 4050-030 Porto, Portugal
b
 Department of Pharmaceutical Technology, ISCSN, Gandra, Portugal
c
 Department of Pharmaceutical Technology, Faculty of Pharmacy of the University of Coimbra, Coimbra, Portugal
Received 2 July 2006; received in revised form 12 September 2006; accepted 13 September 2006Available online 19 September 2006
Abstract
A nanoparticle insulin delivery system was prepared by complexation of dextran sulfate and chitosan in aqueous solution. Parameters of theformulation such as the final mass of polysaccharides, the mass ratio of the two polysaccharides, pH of polysaccharides solution, and insulintheorical loading were identified as the modulating factors of nanoparticle physical properties. Particles with a mean diameter of 500nm and a zetapotential of approximately
15mV were produced under optimal conditions of DS:chitosan mass ratio of 1.5:1 at pH 4.8. Nanoparticles showedspherical shape, uniform size and good shelf-life stability. Polysaccharides complexation was confirmed by differential scanning calorimetry andFouriertransformedinfra-redspectroscopy.Anassociationefficiencyof85%wasobtained.InsulinreleaseatpHbelow5.2wasalmostpreventedupto 24h and at pH 6.8 the release was characterized by a controlled profile. This suggests that release of insulin is ruled by a dissociation mechanismandDS/chitosannanoparticlesarepH-sensitivedeliverysystems.Furthermore,thereleasedinsulinentirelymaintaineditsimmunogenicbioactivityevaluated by ELISA, confirming that this new formulation shows promising properties towards the development of an oral delivery system forinsulin.© 2006 Elsevier B.V. All rights reserved.
Keywords:
Chitosan; Dextran sulfate; Insulin; Nanoparticles; pH-dependent release; Bioactivity
1. Introduction
Colloidal carriers made under mild conditions by complexa-tion of oppositely charged natural polymers, also known aspolyelectrolytes, represent a promising vehicle to protect pro-teins against harsh gastrointestinal conditions and to controlthe release rate of proteins[1–3].Also, natural polysaccha- rides have demanded particular interest due to their attractivebiocompatible, biodegradable, hydrophilic and protecting pro-perties, which have demonstrated favourable characteristics fordrug entrapment and delivery[4].However, due to the labile physicochemical properties of proteins, gentle nanoencapsula-tion conditions must be provided to assure the maintenance of their structural bioactivity. Particular attention has been givento protein-loaded chitosan-based nanoparticles[5–7].Chitosan,
Corresponding author. Tel.: +351 222078949; fax: +351 222003977.
 E-mail address:
an unbranched polyamine of 
d
-glucosamine and
-acetyl glu-cosamine molecules, is characterized for its biodegradable,non-toxic and biocompatible properties[8]providing severalbiomedical, pharmaceutical and food applications. Moreover,chitosan has the significant potential of reducing transepithelialelectrical resistance and transiently opening tight conjunctionbetween epithelial cells[9].In addition, its mucoadhesive prop- erty[10]is another advantage for promoting drug adsorption due to combination with anionic substructures such as sialicacid moieties of the mucosa layer. The adhesion of chitosanat the site of drug absorption offers various advantages for animproved uptake of therapeutic peptides[11,12].Additionalcoincorporationofhighlychargeddensitypolyan-ionsontochitosannanoparticlescanimprovenanoparticleprop-erties concerning protein association efficiency and modulationof drug release[13,14].It has been previously noted that the stronger the protein–polyions complex is the slower will be therateofproteinreleaseduetoenhanceofelectrostaticinteractions[15].
0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.colsurfb.2006.09.012
 
194
B. Sarmento et al. / Colloids and Surfaces B: Biointerfaces 53 (2006) 193–202
Taking into account these considerations, dextran sulfate(DS), a biodegradable and biocompatible branched negativelycharged polyion with approximately 2.3 sulfate groups per glu-cosyl residue, was selected to formulate DS/chitosan nanoparti-cles. Chitosan and DS have been formulated as polyelectrolytecomplex gels with swelling characteristics[16]and controlled release properties[17]and have been extensively used as multi- layer films for cell culture[18].Additionally, they are intrin- sically hydrophilic, which contributes for longer circulationtimes
in vivo
and allows the encapsulation of water-solubleproteins.In the present work insulin was chosen as the model protein.Itisawellknown51aminoacidsprotein,producednowadaysbyDNA recombination techniques and used subcutaneously in thetreatment of diabetes
mellitus
. Although several attempts havebeen developed regarding alternative routes of insulin adminis-tration[19–21],the oral approach remains the most attractive due to convenience and high patient compliance. However, thebioavailability of insulin after oral administration is normallylow, due to its low stability in the gastrointestinal tract (GI),lowpartitioncoefficientandthephysicalbarrieroftheintestinalepithelium.Oneapproachtoimprovethegastrointestinaluptakeoflowmolecularweightproteinsistobindthemtocolloidalsys-temslikenanoparticles,protectingthemfromdegradationinthegastrointestinal tract and promoting the transport into systemiccirculation[22,23].Sincethesizeofnanoparticlesandproteinstabilityareessen-tial for an efficient pharmacologic effect of insulin, the mainobjective of this paper was to establish a nanoparticulate sys-tem produced by polyelectrolyte complexation between DS andchitosan, and evaluate their potential as insulin carriers. Thephysical and morphological properties of nanoparticles wereinvestigated in accordance with formulation parameters and thereleaseprofileofinsulinwasalsodeterminedregardingitspoten-tial for oral delivery.
2. Materials and methods
2.1. Materials
Low molecular weight (MW) chitosan (
50kDa) and lowMW dextran sulfate (8kDa) were purchased from Sigma (Por-tugal). High MW dextran sulfate (500kDa) was obtained fromPKC
®
(Denmark). Dextran sulfate stock solutions were pre-pared in deionized water (Milli-Q
®
) by overnight magneticstirring and chitosan solutions were prepared by dissolutionof chitosan in deionized water containing 1% (v/v) acetic acidfollowed by filtration through a paper filter Millipore #2 andstored at 4
C. Human zinc-insulin crystal (Lot RS0325, 7.0mglyophilized human biosynthetic insulin per vial) was a gift fromLilly Portugal.
2.2. Preparation of nanoparticles
Nanoparticle complexation between DS and chitosan wasperformed employing aqueous solutions of oppositely chargedpolymers in a final volume of 120mL. Unless otherwise men-tioned, complexes were obtained after dropwise addition of chitosan solution at pH 5.0 to DS solution at pH 3.2 undermagnetic stirring followed by additional mixing for 15min at600rpm to final concentrations of 0.15% DS and 0.10% chi-tosan (DS/chitosan mass ratio 1.5:1). For insulin association,7mg of the protein was previously dissolved in the DS solutionbefore chitosan complexation. These conditions were obtainedafter preliminary studies that provided the best results takinginto account the desired mean particle size and insulin associa-tion efficiency.
2.3. Nanoparticle characterization
Measurements of particle size were performed by photoncorrelation spectroscopy (PCS) at 25
C with a detection angleof 90
and zeta potential by laser Doppler anemometry (LDA)using a Malvern Zetasizer and Particle Analyzer 5000 (MalvernInstruments) (
n
6).
2.4. Stability of nanoparticles
The stability of nanoparticles was determined monitorizingtheir mean particle size and zeta potential over time. After pro-duction, nanoparticles were stored at 4
C in aqueous solutionand then the size and zeta potential were measured after 0, 7,14, 28, 42 and 60 days of shelf-life.
2.5. Isolation of particles
Nanoparticleswerecollectedbycentrifugationat14,000rpm(20,000
×
g
)for45min.Supernatantwereusedforinsulindeter-mination. Nanoparticles were kept at 4
C for further experi-ments.
2.6. Insulin association efficiency
Theamountofinsulinassociatedwiththeparticleswascalcu-lated by the difference between the total amount used to preparethe particles and the amount of insulin present in the aqueousphase after centrifugation. The association efficiency (AE) wasdetermined indirectly applying the following equation:AE
=
total amount of insulin
free insulin in supernatanttotal amount of insulin
×
100
2.7. In vitro release studies
Nanoparticleswereplacedinto20mLofUSPXXVIbuffers,namely hydrochloric acid pH 1.2, acetate pH 4.5, acetate pH 5.2and phosphate pH 6.8 and incubated at 37
C under magneticstirring. At pre-determined time intervals, samples were takenfor insulin determination and replaced by fresh medium.
2.8. Insulin determination
Insulin concentration was determined spectrophotometri-cally using the Coomassie PlusTM Bradford Assay (Pierce,
 
 B. Sarmento et al. / Colloids and Surfaces B: Biointerfaces 53 (2006) 193–202
195
Rockford, USA) modified Bradford assay[24].Briefly, insulin samples and Bradford reagent were mixed at 1:1 (v/v) ratio ina 96-well plate and incubated at room temperature for 15min.The absorbance was measured at 595nm on a thermomax platereader (PowerWaveX; Bio-Tek, Winooski, VT, USA). Calibra-tion curves were made using supernatant of unloaded particlesfor AE and unloaded nanoparticles for
in vitro
release profile.All the results were made in triplicate.
2.9. Scanning (SEM) and transmission (TEM) electronicmicroscopy
For the SEM analysis, nanoparticles were mounted on metalstubs using adhesive tape, gold coated under vacuum and exam-ined on a JEOL JSM-840 SEM (Japan). For TEM, one dropof sample was placed in a grid, treated with uranil acetate andobserved in a Zeiss EM 902A TEM.
2.10. Differential Scanning calorimetry (DSC)
Thermograms were obtained using a Shimadzu DSC-50system (Shimadzu, Kyoto, Japan). Samples were lyophilized,crimped in a standard aluminium pan and heated from 20 to350
Catarateof10
C/minunderconstantpurgingofnitrogenat 20ml/min.
2.11. Fourier transform infra-red (FTIR) analysis
IR-spectra were measured using a Bomem IR-spectrometer(Bomem,Canada).Sampleswerelyophilized,gentlymixedwith300mgofmicronizedKBrpowderandcompressedintodiscsata force of 10kN for 2min using a manual tablet presser (PerkinElmer,Norwalk,USA).Foreachspectruma256-scaninterfero-gramwascollectedwitha4cm
1
resolutioninthemid-IRregionat room temperature. Insulin spectra were obtained according toa double subtraction procedure[25]and insulin-free systemsand water vapor spectra were collected under identical condi-tions for blank subtraction. All samples were run in triplicateand the data shows the average of the three measurements.
2.12. Immunological bioactivity of insulin
The integrity of the insulin released from nanoparticles wasassessed by a reported HPLC method[26]while its bioactivitywas assayed by an ELISA test (Mercodia, Uppsala, Sweden).Aliquots of samples taken during release assays were accu-rately diluted with deionized water (Milli-Q
®
) regarding theideal concentration range of the method (1–200mU/l) using themanufacture’s protocol and its relative bioactivity was calcu-lated by comparison with values obtained by Bradford analysisof the same aliquots.
2.13. Statistical analysis
The
-test and the one-way analysis of variance (ANOVA)with the pairwise multiple comparison procedures (Student–Newman–Keuls method) were performed to compare two ormultiple groups, respectively. All analyses were run using theSPSS program (Version 14.0, SPSS Inc., USA) and differenceswere considered to be significant at a level of 
P
<0.05.
3. Results and discussion
Complex coacervation between chitosan and DS is a mildprocess and was used to prepare nanoparticles at ambient tem-perature without using sonication or organic solvents. ResultingDS/chitosan nanoparticle colloidal suspension presented a deepand typical Tyndall effect. Thus, it is a suitable procedure toencapsulate sensitive materials such as proteins, which are sen-sitive to different stress factors[27].Being insulin an ampho- teric molecule it is able to be electrostatically attached to suchnanoparticles composed of DS and chitosan with a high avidity,allowing its association with the stable colloidal drug carrier.The insulin monomer contains many ionizable groups due to6 amino acid residues capable of attaining a positive chargedpolyelectrolyte like chitosan and 10 amino acid residues capa-ble of attaching a negative charged polyelectrolyte like DS[28].Thesepropertiesmightbehighlyresponsiblefortheentrapmentof insulin into DS/chitosan nanoparticles.Factors affecting the characteristics of DS/chitosan nanopar-ticles and the optimal conditions for their preparation werestudied.Primary assays were done in order to establish the range of polyelectrolytes concentration to produce nanoparticles with anappropriate submicron size and a significant value of insulinAE. Therefore, concentrations range from 0.05% to 0.20% forboth chitosan and DS were used to prepare nanoparticles in theproportion of 1:1, at final pH of 4.8. Results are depicted inTable 1.As expected, increasing the polyelectrolytes concentrationled to an increase of nanoparticles mean size in a linear depen-dency.
Table 1Influence of polyelectrolytes concentration on nanoparticle propertiesDS (%) Chitosan (%)
z
-Average number (nm) AE (%) Zeta potential (mV)0.05 0.05 479
±
118
*
54.7
±
4.8
*
0.2
±
0.10.10 0.10 665
±
210 71.5
±
2.9
*
0.4
±
0.10.15 0.15 846
±
100 87.4
±
2.7
*
1.2
±
0.30.20 0.20 1012
±
187
*
96.4
±
4.3
*
3.2
±
0.4The ratio between chitosan and DS was kept at 1/1 (w/w).
*
The mean difference is significant at the 0.05 level.

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